Cancer and other diseases involving abnormal or undesired cellular proliferation present a major challenge to the pharmaceutical industry. Desirable therapeutic compounds frequently act on cellular targets to inhibit cellular growth and/or kill unwanted cells. In order to identify such therapeutic compounds efficiently, it is often desirable to identify the cellular targets that are involved in such growth inhibition or cell death. Yet such cellular targets are difficult to identify, because cells exhibiting the desired phenotype disappear from a cell population, and consequently the targets (and the corresponding causative agents) are lost.
In general terms, experiments that identify agents that inhibit cellular growth and/or kill cells are termed xe2x80x9cnegative selectionsxe2x80x9dxe2x80x94i.e., selections for compounds that exert a cytotoxic or cytostatic effect on a cellular population. Such negative selections are needed for pharmaceutical research relating to a number of areas, including cancer, viral infection and the like. The art to date has not provided efficient, generally applicable methods for conducting negative selections in mammalian cellsxe2x80x94i.e., for directly identifying the causative agents and subsequently recovering the targets that interact with such agents to result in growth inhibition or in cell death.
The lack of efficient negative selection protocols is of particular concern in the field of cancer research. Drug discovery for cancer requires identification of therapeutic agents that interact with endogenous cellular targets so as to provide a cytotoxic effect on the diseased or abnormal cell. Preferably, such agents also will act with specificity for the target cell typexe2x80x94i.e., selectively killing unwanted cells, while sparing healthy, normal cells. One method for identifying such valuable therapeutic agents is to first identify an endogenous cellular target involved in that cytotoxic effect, and then use that target as the basis of a screen to identify small molecule modulators that interact with the target. Alternatively, therapeutic agents may be either proteinaceous compounds that interact with an endogenous cellular target or nucleic acids that prevent either the production or function of that target. In such cases, it is desirable to directly recover the agent that caused the desired cellular inhibition or death.
In the case of cell death, the modulated target may in some instances be involved in an apoptotic pathway, and in other instances, may be involved in necrosis. In general terms, apoptosis is the process of normal, programmed cell death in an organism, while necrosis is a less specific, regulated response that lacks many biochemical features associated with apoptosis. Many clinical manifestations of cancer are believed to represent a malfunction in this normal apoptotic processxe2x80x94i.e., a failure of normal cell death, leading to uncontrolled proliferation of transformed cancer cells in the body. Thus, the pharmaceutical industry particularly desires to identify agents that will selectively promote the apoptotic process, thereby encouraging death of the unwanted cancerous cells.
Much of the current research for new chemotherapeutic agents focuses largely on identifying new compounds that interact with, or modulate the effect of, proteins that are already known to play a key role in a given disease pathway. One such example is recent work on the role of thymidine kinase in cancer, and the resulting discovery of 5-Fluorouracil and folate analogues. Such techniques, however, are inherently limited by the scope of pre-existing knowledge of such key proteins. To maximize the development of new chemotherapeutic agents for, e.g., cancer, it is preferable to be able to broadly and generally screen for cytotoxic compounds without being so limited to a small pre-existing pool of targets.
Several general methods relate to the identification of dead or dying cells, but lack the ability to directly identify substances that caused the cell death (and, therefore, do not lead to the direct identification of the cellular target that modulates its cytotoxic effect); for example, a variety of staining methods identify necrotic and/or apoptotic cells. Such methods include antibody staining techniques and dye staining techniques such as, e.g., propidium iodide staining. Other assays employ laborious replica plating techniques, whereby duplicate colonies are established and one such colony is exposed to putative cytotoxic agents. When cellular death is observed in the one colony (via its death, or absence from a replica plate), its corresponding duplicate is then subjected to further analysis. However, such replica plating techniques are time-consuming and not suited to high-throughput screening procedures. Moreover, at best the replica plating technique is an approximation, as the actual endogenous cellular materials that are involved in the cell death are lost with the duplicate colony that disappears from the replica plate.
Thus, a need exists for a negative selection technique that is direct (i.e., it is the dead or dying cells themselves that provide the causative agents and corresponding endogenous targets relating to their death). Moreover, a need exists for a negative selection technique that provides rapid, efficient evaluationsxe2x80x94i.e., a technique that is suitable for high-throughput screening. The present invention meets these needs.
The present invention provides methods for performing negative selections. In some embodiments, the negative selections are performed by introducing a genetic library into a population of target cells, collecting a subpopulation of cells that disattach from a culturing surface, and then recovering the genetic material from that subpopulation. In other embodiments, the invention provides methods for obtaining cytotoxic agents that establish a lethal phenotype, wherein a genetic library is introduced into a population of target cells, a subpopulation of cells displaying a lethal phenotype is collected, and genetic material is then recovered from that subpopulation. In variations of these embodiments, cell-specific cytotoxic agents are identified by employing a counterscreening step wherein the genetic material from the subpopulation displaying disattachment and/or the lethal phenotype is introduced into a second, different population of cells, and a second sublibrary of genetic material is obtained from a second subpopulation that does not display disattachment and/or the lethal phenotype.
A variety of particular embodiments exist for each of these basic embodiments. In some particular embodiments, the lethal phenotype of the methodology may be apoptosis, necrosis, or growth arrest. In embodiments in which the lethal phenotype is apoptosis, the property of disattachment from a culturing substrate may be used as a surrogate for apoptosis, thereby providing a technique for enriching the apoptotic cell population. In other particular embodiments, the genetic material may be partially sequenced, or the method steps may be reiterated in a second population of the same cells. The target cells may be mammalian cells, or more particularly primary cells, especially primary cells derived from epithelial or endothelial cells, stem cells, mesenchymal cells, fibroblasts, neuronal cells or hematopoeitic cells. The mammalian cells may also be cancer cells, or more particularly cancer cells that are metastatic or derived from solid tumors. The cancer cells may particularly be derived from breast, colon, lung, melanoma or prostate tissue. In other particular embodiments, the mammalian cells are genetically altered, and more particularly may be immortalized or transformed.
In embodiments that utilize the property of disattachment of target cells from a culturing surface, particular embodiments will feature a low background of spontaneously disattaching cells, which may more particularly be no more than about 10%, or alternatively no more that about 2%. Target cells having such low backgrounds include SW620 and HT29 colon cancer cells, T47D breast cancer cells, and HuVEC cells. In particular embodiments, the disadhering cells are collected over a period of at last about 12 hours. In still other particular embodiments of the basic embodiments, the genetic library is large or even very large(xcx9c105 encoded putative cytotoxic agents).
The invention also encompasses the identification of small organic molecules that induce a lethal phenotype. In some embodiments, organic molecules that displace a proteinaceous cytotoxic agent from an endogenous protein are obtained. In other embodiments, organic molecules having a structure-activity relationship with that proteinaceous cytotoxic agent are identified.
The invention also lends itself to embodiments that screen for conditional cytotoxicity, wherein a genetic library is introduced into a population of target cells, exposing those target cells to a subtoxic threshold dose of a secondary reagent, collecting a subpopulation of cells displaying a lethal phenotype, and recovering genetic material from that subpopulation. Again, in particular embodiments the lethal phenotype may be apoptosis, necrosis or growth arrest. In other particular embodiments, the secondary reagent may be UV, X-ray or neutron radiation, or may be a chemotherapeutic agent, more particularly methotrexate, cisplatin, 5-fluorouracil, colchicines, vinblastine, vincristine, doxyrubicin or taxol. Particular embodiments include cancer cells, more particularly solid tumors, as target cells, counterscreening with a second cytotoxic substance, preconditioning the target cells prior to exposure with, e.g., growth factors, cytokines, chemokines, or activation of oncogenes.
The invention also encompasses compositions of matter, more particularly six representative amino acid sequences, that are obtained by applying the inventive negative selection methods to HT29 colon cancer cells.